Spermatogonial stem cell technologies: applications from human medicine to wildlife conservation†

Abstract

Spermatogonial stem cell (SSC) technologies that are currently under clinical development to reverse human infertility hold the potential to be adapted and applied for the conservation of endangered and vulnerable wildlife species. The biobanking of testis tissue containing SSCs from wildlife species, aligned with that occurring in pediatric human patients, could facilitate strategies to improve the genetic diversity and fitness of endangered populations. Approaches to utilize these SSCs could include spermatogonial transplantation or testis tissue grafting into a donor animal of the same or a closely related species, or in vitro spermatogenesis paired with assisted reproduction approaches. The primary roadblock to progress in this field is a lack of fundamental knowledge of SSC biology in non-model species. Herein, we review the current understanding of molecular mechanisms controlling SSC function in laboratory rodents and humans, and given our particular interest in the conservation of Australian marsupials, use a subset of these species as a case-study to demonstrate gaps-in-knowledge that are common to wildlife. Additionally, we review progress in the development and application of SSC technologies in fertility clinics and consider the translation potential of these techniques for species conservation pipelines.

Keywords: in vitro spermatogenesis; male infertility; marsupials; spermatogonial stem cells; spermatogonial transplantation; stem cell technologies; testis grafting; wildlife conservation.

Graphical Abstract

Figure 1

Spermatogonia population dynamics in mouse, human and marsupials. (A) In rodents, the revised A_single model illustrates germline stem cell dynamics, where a subset of A_single spermatogonia constitutes the SSC population. Transitioning into a progenitor state, these undifferentiated spermatogonia undergo incomplete cytokinesis, forming pairs (A_paired) and chains (A_aligned). Progenitors commit to differentiation in response to retinoic acid, transitioning into A1, then A2, A3, A4, Intermediate and Type B spermatogonia through successive mitotic divisions, before entering meiosis at the spermatocyte stage. Haploid spermatids resulting from meiosis undergo morphological changes during spermiogenesis to ultimately form mature spermatozoa. (B) In humans (A_pale/A_dark model), undifferentiated spermatogonia may undergo one or two successive mitotic divisions as progenitors or transition directly into Type B differentiating spermatogonia upon the retinoic acid pulse without clonal expansion. (C) Much remains unknown about SSC dynamics in marsupials (designated with question marks). It is unclear if undifferentiated type A spermatogonia undergo transit-amplifying divisions before transitioning into Intermediate and Type B differentiating spermatogonia. Spermiogenesis in several Australian marsupials exhibits unique morphological changes in the haploid spermatids, differing from placental animals.

Figure 2

Comparison of single cell RNAseq data from human, mouse and opossum SSCs. (A) Venn diagram depicts the number of shared and unique differentially expressed genes in human and mouse undifferentiated spermatogonia (Hermann et al., 2018), and opossum spermatogonia (Murat et al., 2023). Thirty-two genes were common across all three datasets, representing putative conserved markers of SSCs between these species (also see Supplemental Dataset S1). (B) Conserved biological processes between eutherian and marsupial spermatogonia identified by gene ontology analysis (see Supplemental Dataset S1 for complete analysis).

Figure 3

Growth factor signaling in the SSC niche. (A) The SSC niche is an “open” niche consisting of spermatogonia and surrounding somatic support cells, including Sertoli, Leydig, and PTM cells. The somatic cells release growth factors to promote maintenance and self-renewal of SSCs (arrows) including Sertoli cell-derived GDNF and FGF2; CSF1 from Leydig cells, myoid cells and interstitial macrophages; and IGF1 from Leydig cells. (B) GDNF binds to the GFRA1/RET receptor complex to activate multiple signaling cascades, including the PI3K/Akt pathway via SRC kinase recruitment, leading to N-myc expression and SSC proliferation. Concurrently, it triggers rapid and transient Ras/MAPK pathway activation, upregulating cell cycle activator genes (Cdk2, c-fos, cyclin A). FGF2 binding to FGFRs induces receptor tyrosine kinase dimerization and phosphorylation, activating various pathways (PI3K/Akt, Ras/MAPK, PLCγ/PKC, JAK/STAT, Wnt/β-catenin), modulating genes involved in undifferentiated spermatogonia survival, growth, and proliferation. The IGF1-IGF1R interaction also activates the PI3K/Akt pathway, enhancing SSC survival and stem cell capacity. SCF1R activation by CSF1 also promotes SSC self-renewal and proliferation. CDH2 and INTGa6/b1 are important for cell–cell and cell-extracellular matrix interactions that are crucial for SSC function. Cell surface markers, signaling pathways and genes that we found to be conserved between the opossum, mouse and/or human are indicated in red. Figure created using BioRender.

Figure 4

Overview of conservation pipelines using SSC biobanking. The proposed conservation pipelines for SSC biobanking begin with the collection and cryopreservation of testicular tissue or isolated SSCs from recently deceased endangered or vulnerable animals. Following expansion in culture, cryopreserved-thawed or fresh SSCs could be transplanted into the testis of another individual of the same species (allogeneic) to propagate donor genetics through natural mating. Alternatively, donor SSCs could be transplanted into a recipient from a closely related species (xenotransplantation) to facilitate in vivo spermatogenesis and sperm recovery for assisted reproduction approaches. Stored testis tissue could also be grafted onto an individual of the same species (allograft) or different species (xenograft), functioning as a bioincubator to facilitate ex vivo spermatogenesis and sperm recovery. Additionally, isolated SSCs or thawed testicular tissue could be cultivated in an environment conducive to stem cell differentiation and sperm production through in vitro spermatogenesis. The retrieved sperm could then be utilized via ICSI. Figure created using BioRender.